|dc.description.abstract||The current, grim world-TB scenario, with TB being the single largest infectious disease
killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium
tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003).
This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited.
The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development.
In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase.
Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays
described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact
that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded.
The availability of only three crystal structures for a critical enzyme like
dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of
dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter.
It is interesting to note that the P-loop-containing nucleoside monophosphate kinases
(NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches.
As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom.
Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.||en_US